Attenuated and adapted strain of pseudomonas for delivering antigens

ABSTRACT

A process for obtaining an adapted strain of  Pseudomonas  includes the following steps: deleting the genes ExoS, ExoT, aroA and lasl in an initial  Pseudomonas  strain cultivated in a LB medium; progressively cultivating this strain in a chemically defined medium based on a glucose minimal medium supplemented with magnesium and calcium; wherein the adapted strain presents the same toxicity and secretion capacities than the initial strain, and its doubling time when cultivated in the chemically defined medium is less than 60 minutes. An adapted strain is furthermore treated to become “killed but metabolically active.”

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.14/364,322, filed on Jun. 11, 2014, which is a National Phase Entry ofInternational Application No. PCT/EP2012/075167, filed on Dec. 12, 2012,which claims priority to European Patent Application No. 11306636.9,filed on Dec. 12, 2011, all of which are incorporated by referenceherein.

FIELD

The present invention concerns a method for obtaining an attenuated andadapted strain of Pseudomonas for its use in human vaccines. Theinvention also concerns the obtained strain, and the other treatmentsuseful to improve the safety of the Pseudomonas strain, withoutdecreasing its immunogenicity.

BACKGROUND

Bacteria have been developed for use as vaccines that deliverheterologous antigens. These bacteria have been modified to containnucleic acid sequences encoding a protein or antigen of interest.However, injection of native virulent infectious bacteria is potentiallydeleterious to the recipient organism. Therefore, virulence of bacteriahas to be attenuated before their use in immunotherapy.

Due to the endowed effective ability to deliver antigen to antigenpresenting cells in vivo, type III secretion system (T3SS or TTSS) basedattenuated bacterial vectors attracted more and more attention for theirpotential use, in particular for cancer vaccine development. In the lastdecades, the use of Gram-negative bacteria, such as Salmonella,Shigella, Yersinia and Pseudomonas which use their powerful secretionmachinery—type III secretion system to deliver bacterial effectors tothe membrane or into the host cell cytoplasm, has attracted more andmore attention for their potential use for cancer vaccine development(Epaulard et al., 2006). Until now, T3SS based bacteria has been provedas a carrier for cancer vaccines which provide the protection againstseveral tumor models like glioma, prostate cancer, breast cancer andfibrosarcoma in mice.

Beside their high efficiency, the safety of bacterial vectors are veryimportant for clinical application. In our previous work described inthe article from Epaulard et al., published in 2008, two attenuatedstrains of Pseudomonas were described:

one attenuated strain “CHA-OST”, in which the exoS exoT genes encodingtwo major T3S toxin exoenzymes—ExoS and ExoT—have been deleted,demonstrated efficient antigen delivery ability and tumor protectionperformance;

a more attenuated strain “CHA-OAL”, in which beside exoS and exoT genes,the aroA and lasl genes have also been deleted. The aroA gene encodesthe 3-phosphoshikimate 1-carboxyvinyltransferase which is a key enzymein aromatic amino acid synthesis and the last gene encodes the enzymewhich produces quorum sensing (QS) homoserine lactones 3-oxo-C12-HSL.

This second strain CHA-OAL has a greatly reduced toxicity while keepinga good efficiency at high doses. This maximally attenuated strain mayrepresent the best compromise between virulence attenuation andefficiency so that it was endowed the potential for clinicalapplications.

The patent EP 1 692 162 describes an expression vector of chimericproteins, comprising the functional part of the proteins ExoS or ExoTfrom Pseudomonas aeruginosa, and an antigen of interest. The chimericprotein can be expressed by a strain transformed with said vector, andbe “injected” into the cytoplasm of an antigen-presenting cell via theTTSS system. Preferentially, the transformed strain is a CHA-OST strainwherein the genes exoS and exoT have been deleted.

Recently, a new concept of vaccines that are “killed but metabolicallyactive” (KBMA) have been described. They retain the immunologicalproperties of live organisms but have a safety profile closer to that ofkilled organisms. Initially, the KBMA vaccine strategy was demonstratedby Brockstedt et al. with Listeria monocytogenes bacteria (Brockstedt etal., 2005). The deletion of two uvr genes (A and B) coding forexonucleotidase A and B subunit renders bacteria sensitive topsoralen-induced crosslink by exposure to long wavelength UVA light(Wollowitz, 2001). However, it is unclear whether this photochemicaltreatment could be applied to Pseudomonas bacteria and whether the typeIII secretion system would work after this treatment.

For clinical purpose, it is also important to join good manufacturingpractices for vaccine production. In particular, bacterial vectorsshould be produced in chemically defined medium with constant growthperformance to ensure the quality of the product.

Indeed, one of the main technical problem met with the culture ofattenuated Pseudomonas strains is their poor growth rate in standardchemically defined growth media. In recent studies, one chemicallydefined medium—glucose minimal (M9) medium has been applied in differentbacterial species culture, such as E. Coli, Salmonella Typhimurium,Pseudomonas putida, for the investigations of genes expression, proteinexpression and bacterial communities. This medium was previouslydeveloped by DeBell R M and had been proved to be ideal for Exotoxin Aproduction by P. aeruginosa (DeBell, 1979).

There is a need in the art for an optimized chemically defined medium,allowing a high and constant growth rate for Pseudomonas attenuatedstrains. Preferentially, these strains are transformed with a vector ofexpression such as described in patent EP 1 692 162.

SUMMARY

The present invention is related to a process for obtaining an adaptedstrain of Pseudomonas, comprising the following steps:

Deleting the genes ExoS, ExoT, aroA and lasl in an initial Pseudomonasstrain cultivated in a LB medium,

Progressively cultivating this strain in a chemically defined mediumbased on a glucose minimal medium (M9) supplemented with magnesium andcalcium,

wherein the adapted strain presents the same toxicity and secretioncapacities than the initial strain, and its doubling time whencultivated in said chemically defined medium is less than 60 minutes.

In a preferred aspect of the invention, the Pseudomonas strain isfurthermore treated with the KBMA process to become “killed butmetabolically active”. The invention is also related to a “killed butmetabolically active” Pseudomonas strain. The present invention is alsorelated to a Pseudomonas strain, such as obtained by the processpreviously described, characterized by a better growth in the chemicallydefined medium than in LB medium, with a similar toxicity and a similaractivity of the T3SS injection system than the strain before adaptation.

According to the present invention, the attenuated adapted Pseudomonasstrain expresses a chimeric protein composed of the N-terminal sequenceof ExoS or ExoT, and an antigen of interest. The invention is alsorelated to immunogenic compositions and vaccines comprising thepreviously described strains. Finally, the invention also concerns avaccination strategy characterized by multi-position injections of theattenuated and adapted strain of Pseudomonas, to the organism.

DETAILED DESCRIPTION

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors which arereported in the publications and which might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional microbiological and molecularbiological techniques within the skill of the art. Such techniques arewell known to the skilled worker, and are explained fully in theliterature. See, for example, Sambrook et al., 2001. Conventionalimmunological techniques are explained in Current protocol inImmunology, Coligan, John Wiley & Sons (2005).

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “astrain” includes a plurality of such strains, and so forth. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any materials and methodssimilar or equivalent to those described herein can be used to practiceor test the present invention, the preferred materials and methods arenow described.

The present invention is related to a process for obtaining an adaptedstrain of Pseudomonas, comprising the following steps:

Deleting the genes ExoS, ExoT, aroA and lasl in an initial Pseudomonasstrain cultivated in a LB medium,

Progressively changing the culture medium from LB medium to a chemicallydefined medium comprising magnesium and calcium, and

Selecting the adapted strains presenting a doubling time inferior to 60minutes when cultivated in said chemically defined medium.

Advantageously, the adapted strain presents the same toxicity andsecretion capacities than the initial strain.

As used herein, the following terms may be used for interpretation ofthe claims and specification. In the claims which follow and in thepreceding description of the invention, except where the contextrequires otherwise due to express language or necessary implication, theword “comprise” or variations such as “comprises” or “comprising” isused in an inclusive sense, i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

The term “Pseudomonas” designates a genus of gammaproteobacteria,belonging to the family Pseudomonadaceae containing 191 validlydescribed species. All species and strains of Pseudomonas areGram-negative rods, and are classified as strict aerobes. Among them,Pseudomonas aeruginosa is a highly relevant opportunistic humanpathogen.

Gram-negative bacteria use different types of secretion systems fortheir own purposes. In particular, the type III secretion system (T3SS)is involved in the cytotoxicity of Pseudomonas strains.

The first step of the process according to the invention is the deletionof the genes ExoS, ExoT, aroA and lasl in an initial Pseudomonas straincultivated in a LB medium. The exoS and exoT genes encodes two major T3Stoxin exoenzymes—ExoS and ExoT. The aroA gene encodes the3-phosphoshikimate 1-carboxyvinyltransferase which is a key enzyme inaromatic amino acid synthesis and the lasl gene encodes the enzyme whichproduces quorum sensing (QS) homoserine lactones 3-oxo-C12-HSL. Aspreviously described in (Epaulard et al., 2008), the obtained strainpresent an attenuation of its virulence while keeping a good efficiencyto induce an immunogenic response, when administered to an organism.

Luria Bertani (LB), a nutritionally rich medium, is primarily used forthe growth of bacteria. The LB medium is widely used as a growth mediumfor all types of bacteria, and its composition is well known by the manskilled in the art.

The second step of the process according to the invention is aprogressive change of growth medium, from LB medium to a chemicallydefined medium based on a glucose minimal medium (M9) supplemented withmagnesium and calcium, named MM9 medium. Advantageously, the replacementof LB broth by the MM9 medium was made according to the followingproportions: 100% LB->50% LB-MM9->20% LB-MM9->5% LB-MM9->2% LB-MM9->100%MM9. For each step, at least two days of adaptation were realized, untilthat the adapted strain proliferates stably in 100% MM9 medium.

The third step of the process according to the invention is theselection of adapted strains, characterized by a growth capacity definedby a “doubling time” than is less than 60 minutes. The “doubling time”is the necessary period for a bacterial colony to double its populationwhen cultivated in optimal conditions (37° C. in a medium growth, underconstant moving).

The final product of the process according to the invention is an“adapted strain” presenting the following features: the adapted strainpresents the same toxicity and secretion capacities than the initialstrain, while its doubling time when cultivated in said chemicallydefined medium is less than 60 minutes. Advantageously, its doublingtime in said chemically defined medium is less than 50 minutes, and morepreferentially is less than 45 minutes.

In a specific aspect of the invention, the MM9 medium is a M9 mediumsupplemented with 1 mM Mg²⁺ and 1 mM Ca²⁺. Advantageously, thechemically defined medium MM9 has the following composition:

TABLE 1 Composition of modified M9 (MM9) medium Extract of syntheticyeast   4 g/L Tryptophan 1 mmol/L (0.2 g/L) Glucose 14 mmol/L  (2.5 g/L)Glycerol 1% FeSO4 0.4 g/L Citric acid 2 mmol/L (0.36 g/L)  M9 SaltsMedium 5X (Sigma) approximate composition per liter Na2PO3 anhydre 33.9g KPO3 15.0 g NaCl  2.5 g NH4Cl  5.0 g MgSO₄ 1 mmol/L (50 mg/L) CaSo₄ 1mmol/L (50 mg/L) Eau qsp 1 L

In a specific aspect of the invention, the adapted strain is furthermoretreated to become “killed but metabolically active”. The process toobtain killed but metabolically active bacteria can be summarized asfollow:

A Pseudomonas strain is deleted for the gene uvrAB; any technique knownby the man skilled in the art can be used to obtain this deletion;

A photochemical inactivation with UVA is performed in presence of S-59psoralen;

Toxicity, antigen production and antigen secretion via the TTSS systemby the treated strain are assessed.

For more details on the KBMA process, see (Brockstedt et al., 2005) and(Wollowitz, 2001), herein incorporated by reference. In a preferredaspect of the invention, the obtention of a KBMA P. aeruginosa strain ismade in presence of a concentration of 10 μM of S59-psoralen with an UVAirradiation at a dose of 7.2 J/cm².

The invention is also related to a process such as described above, toobtain a Killed But Metabolically Active Pseudomonas strain, and to theobtained strain. In particular, the KBMA process can be applied to a“CHA-OST” or to a “CHA-OAL” strain. The invention is also related to aKBMA Pseudomonas strain, non-toxigenic and less virulent than theinitial non-treated strain, while keeping T3SS secretion capacities. Ina specific aspect of the invention, the Pseudomonas strain has beenattenuated prior the KBMA treatment, by the deletion of the genes exoSand exoT.

In another aspect of the invention, the Pseudomonas strain has beenattenuated prior the KBMA treatment, by the deletion of the genes exoS,exoT, aroA and lasl. In another aspect of the invention, the Pseudomonasstrain has been adapted on a MM9 medium prior the KBMA treatment. Inanother aspect of the invention, the Pseudomonas strain has beenattenuated by the deletion of the genes exoS, exoT, aroA and lasl andadapted on a MM9 medium, prior the KBMA treatment.

The invention is also related to a Pseudomonas strain such as obtainedby anyone of the processes described above, wherein the adapted strainpresents the same toxicity and secretion capacities than the initialstrain, and its doubling time when cultivated in said chemically definedmedium is less than 60 minutes. In a particular embodiment of theinvention, the strain expresses a chimeric protein composed of theN-terminal sequence of ExoS or ExoT, and an antigen of interest. Suchstrain was previously described in the patent EP 1 692 162, which isincorporated herein by reference.

In a preferred aspect of the invention, the strain belongs to thespecies Pseudomonas aeruginosa. The invention is also related to aspecific adapted strain of Pseudomonas that has been deposited at theNational Collection of Microorganisms Cultures (CNCM, Pasteur Institute(25 rue du Docteur Roux, 75724 Paris) on Dec. 1, 2011 under theaccession number CNCM I-4564. The invention is also related to animmunogenic composition comprising one of the adapted strain ofPseudomonas such as described above.

The invention is also related to a vaccine comprising the immunogeniccomposition comprising one of the adapted strain of Pseudomonas such asdescribed above and a pharmaceutically acceptable carrier or adjuvant.The man skilled in the art knows the best adjuvant for each vaccinecomposition.

The present invention is also related to a method of inducing an immuneresponse in a host comprising administering to the host by injections inmultiple positions an effective amount of the vaccine such as describedabove. In a particular aspect of the invention, the method of inducingan immune response is based on a vaccination protocol comprising foursubcutaneous injections in right and left flanks of a sufficient amountof the vaccine such as described above (see examples for more details).

The therapeutic use of the adapted and/or KBMA strain of the inventiondepends on the antigen being expressed by the strain. Indeed, the immuneresponse shall be induced against a specific pathogen or tumor.Depending on the disease that has to be treated, a specific antigen ofinterest will be chosen to be expressed as a fusion protein by thePseudomonas strain according to the invention. In a specific aspect ofthe invention, the antigen of interest is a tumoral or a viral antigen.In particular said antigen can be chosen among the list of: BAGE, CAMEL,CEA, DAM, GAGE, HER-2/neu, MAGE, MUM, MART, PSA, PSMA, RAGE, SAGE, andWT1. Preferentially, the antigen is chosen among the tumoral antigenslisted in (Novellino et al., 2005) and (Buonaguro et al., 2011).Finally, the invention is also related to the use of an adapted strainof Pseudomonas such as described above, for preparing a vaccinecomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Evaluation of T3SS-mediated fusion protein secretion by MM9cultivated CHA-CLIN1 strain. The positions of popB (top arrow) and popD(bottom arrow) are marked with arrows; the positions ofS54-PADRE-antigen fusion proteins are marked with stars.

FIG. 2: Photochemically treated P. aeruginosa ΔuvrAB mutant is “killedbut metabolically active”. A. Viability of OST (wt) and OSTAB aftertreatment with the indicated S59 concentration based on colony formingunit on nutrient agar. Mean of 5 experiments realized in duplicatesanalyze with graphpad software. B. Analysis of P. aeruginosa viabilityafter different treatment by Live/Dead BacLight staining kit (Molecularprobe) and measurement of fluorescence in microplate reader. C.Detection by western blot of ExoS54-OVA in supernatant of P. aeruginosaphotochemically treated with different level of amotosalen.

FIG. 3: A. KBMA P. aeruginosa photochemically treated with 10 μMamotosalen produces, secretes and inject antigen into dendritic cells.A. KBMA P. aeruginosa secrete ExoS54-Ovalbumin antigen into thesupernatant. B. The type III secretion system of KBMA P. aeruginosa isfunctional. Injection of S54-Bla in human HL60 cells.

FIG. 4: KBMA Pseudomonas aeruginosa is less cytotoxic. A) Loss ofcytotoxicity was assessed by infection of mouse dendritic cells (BMDCs).LDH release of infected cells was measured at 3 h post-infection. Thepercentages of cytotoxicity were calculated according to the release ofLDH activity. Data are the means of results of at least threeexperiments. B) Cytotoxicity of KBMA P. aeruginosa (PCT 10 μM) to moDCs3 h post infection. C) Effect of MOI on 7AAD/AnnexinV moDCs staining 24h post infection. NT Control represents cells only.

FIG. 5: Vaccination protocols. Evaluation of CHA-CLIN1 strain efficacyby tumor challenge on optimized vaccination protocol. (A) Prophylacticassay. (B) Therapeutic assay.

EXAMPLES

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary.

Example 1. Adaptation of an Attenuated Pseudomonas Strain “CHA-OAL”Cultivated in LB Medium, into a Chemically Defined Medium (MM9)

We firstly adapted CHA-OAL strain in Debell M R modified M9 medium whichwas ideal for Pseudomonas ExoA protein production, but the growth rateof bacteria was not high enough. We further modified M9 medium bysupplementing it with Mg2+ and Ca2+ and the composition of the modifiedM9 medium (MM9) is shown in table 1. In order to adapt the CHA-OALstrain in this medium, the replacement of LB by MM9 for CHA-OAL strainculture was progressive. At the end CHA-OAL strain stably proliferatedin MM9 medium and the growth was much better than it was cultivated inLB broth (table 2).

TABLE 2 Growth kinetic (doubling time) of mutants CHA-OAL in LB and inMM9 broths after adaptation Mean doubling time (min) during exponentialgrowth Mutant LB broth MM9 broth CHA-OAL 88 43This new strain adapted for growing in MM9 medium, with a doubling timeof 43 minutes, was named CHA-CLIN1, and was deposited at the CNCM underaccession number 1-4564.

Example 2. Toxicity and Secretion Capacities of the Adapted StrainCHA-CLIN1 Obtained in Example 1

2.1. Toxicity: In order to verify if the adaptation and the new growthconditions have modified the toxicity of CHA-CLIN1 strain, we assessedthe in vivo toxicity of CHA-CLIN1 strain cultivated in LB or MM9 mediumby observing mortality after one subcutaneous injection of 10⁷, 10⁸, or10⁹ bacteria to 6-week-old female C57BL/6 mice. The toxicity of the newCHA-CLIN1 strain was compared with the toxicity of CHA-OST and CHA-OALstrains, previously described (table 3).

TABLE 3 In vivo toxicity test of CHA-OST and CHA-CLIN1 mutants MutantDose Mortality CHA-OST 10⁵ 0/6 (in LB) 10⁶ 0/6 10⁷ 4/6 CHA-OAL 10⁷ 0/6(in LB) 10⁸ 0/6 10⁹ 0/6 CHA-CLIN1 10⁷ 0/6 (in MM9) 10⁸ 0/6 10⁹ 0/6It can be observed that the toxicity of CHA-CLIN1 strain in MM9 is verylow compared to the toxicity of CHA-OST strain, and is the same than thetoxicity of CHA-OAL strain, cultivated in LB medium.

2.2. Capacity of protein Secretion by TTSS: We tested if T3SS charactersof CHA-CLIN1 strain were modified by both adaptation and growth in MM9medium, compared to a non-adapted strain grown in LB. We transformedCHA-OAL and CHA-CLIN1 strains with pEAI-S54-PADRE-OVACter (transformedstrains are OAL-P-OVA and CLIN1-P-OVA, respectively) and assessed thesecretion of the fusion protein in LB and MM9 medium.

Bacterial cultures for OST-EI (negative control strain), OAL-P-OVA andCLIN1-P-OVA strains were realized at 37° C. with shaking at 250 rpm.After an overnight pre-culture in LB containing 300 μg/mL carbenicillin,the bacteria were resuspended at 0.2, OD₆₀₀ in LB containing 300 μg/mLcarbenicillin, 0.5 mM IPTG, 5 mM EGTA and 20 mM MgCl₂ until the OD₆₀₀reaches a value between 1.5 and 2. Then, bacterial cultures werecentrifuged at 17000 g for 15 min and the supernatant was recovered. Forprecipitation of proteins, perchloric acid was added to supernatant at afinal concentration of 15% and incubated at 4° C., overnight. The nextday, precipitated proteins were centrifuged at 17000 g, 4° C. for 30min; proteins were washed two times with acetone (17000 g, 15 min),dried at room temperature and resuspended in 60 μL denaturation buffer(Tris HCL, dithiothreitol, SDS, bromophenol blue, glycerol).

Then, proteins were analyzed by SDS-PAGE, in a 15% polyacrylamide gel(Ready Gels Recast Gel(Biorad®)), under 120 V, in Tris Glycine SDSmigration buffer (Biorad®), and visualized by Coomassie Blue staining.The secretion results of the different strains are shown in FIG. 1. Inthis figure, the first lane presents strain OST-EAI which is the controlstrain that contains no antigen coding sequence. Three proteins arevisualized:

PopB and PopD (marked with arrows), two structural proteins of T3SS thatform the translocation channel: their presence is a positive control ofa correct activation of T3SS;

the fusion protein P-OVA (marked with stars), observed for thetransformed strains expressing the antigen cultivated both in LB mediumand adapted in M9M medium.

Therefore, the TTSS secretion capacities of CHA-CLIN1 strain in MM9 arethe same than the capacities of CHA-OAL strain cultivated in LB medium,after transformation with a plasmid pEAI-S54-PADRE-OVACter.

Example 3. Attenuation of a Strain “CHA-OST” by the KBMA Process

The nucleotide excision repair mutants OSTΔuvrAB (OSTAB) were generatedfrom the P. aeruginosa strain CHA-OST by Cre/lox-based mutagenesis{Quenee, 2005 #48} and sacB-based negative selection system. S59psoralen/UVA inactivation of bacteria and viability assay. We determinedthe relative sensitivity to photochemical inactivation of OSTAB strainand its parental strain OST over a range of S-59 psoralen concentrationsand an UVA dose of 7.2 J/cm2, using conditions established previouslyfor gram negative bacteria {Lankowski, 2007 #162}.

For photochemical treatment (PCT), the OST ΔuvrAB strain was cultivatedin LB medium at 37° C. until OD₆₀₀ of 0.5. Different concentrations ofS-59 psoralen were added and cultures were grown for an additional hour.1 ml of culture per well (OD₆₀₀ of 1) was then transferred in 6-wellsculture plate for UVA irradiation at a dose of 7.2 J/cm² in aStratalinker 1800 device (Stratagen). Viability of photo-chemicallyinactivated cultures was assessed by serial dilution and plating on PIAfor colony forming units. Points represent mean values of triplicatesplates counted at the most appropriate dilution (FIG. 2A).

As expected, the OSTΔuvrAB strain was much more sensitive to PCT thanOST. With 10 μM S-59 there was ˜1 live replicating organism/1.25×10⁸bacteria exposed for OSTAB, and 2.5×10⁶ live bacteria for OST. Based onthe Live/Dead BacLight Bacterial Viability kit (Molecular Probes), wethen looked for the membrane integrity of the photochemical treated P.aeruginosa strain (FIG. 2B). Under the photochemical treatment, bacteriaare unable to reproduce in nutrient medium but have intact membranes yetthese may be scored as “alive”. Therefore, the present example show thatPseudomonas strains can be subjected to the KBMA treatment, and become“killed but metabolically active”, with intact membranes but incapacityto grow.

Example 4. Secretion Capacities of the KBMA Strain Obtained in Example3: OSTΔuvrAB Mutant is Inactivated by Photochemical Treatment, but StillSecrete Proteins

As shown in FIG. 2C, the fusion protein ExoS54-OVA is detected insupernatant of P. aeruginosa photochemically treated with differentlevel of amotosalen (0, 2, 5, 10, 15 and 200). OSTΔuvrAB was transformedwith plasmid pEAiS54 PADRE Ova. Resulting strains were grown in LBmedium containing 300 mg/L carbenicillin (Cb) during the photochemicaltreatment. Cells were resuspended in LB 300 mg/L Cb with or without 5 mMEGTA and cultivated 3 hours at 37° C. The calcium depletion induced byEGTA triggers P. aeruginosa TTSS activation and secretion of TTSSeffectors in culture medium {Epaulard, 2006 #25}. Supernatants wereprecipitated and analysed by SDS PAGE (Mini protean Tris Glycine 12%precast gel (Biorad)) and immunoblotting with polyclonal rabbit antibodyanti chicken ovalbumin (AbDserotec) used at 1/5000 in TBS BSA 0.5 mg/mLto test the presence of secreted fusion protein ExoS54-PADRE Ova.

FIG. 2C shows that the antigen “PADRE Ova” is secreted as soon as EGTAis added in the culture medium, in any concentration of amotosalen. Asshown in FIG. 3A, KBMA P. aeruginosa photochemically treated with 10 μMamotosalen secretes ExoS54-Ovalbumin antigen into the supernatant.

First, we obtained transformants of OSTΔuvrAB with plasmids pEAI S0-OVA(deleted for secretion Tag) and pEAi S54-OVA. We prepared photochemicaltreated strain in presence of psoralen (0 and 10 μM). Then, we assessedthe production and secretion by TTSS of ExoS54-fused proteins by the PCTstrains. After PCT, we used four growth conditions in LB medium: no TTSSstimulation, TTSS stimulation by calcium depletion (5 mM EGTA), OVAintrabacterial production without TTSS stimulation (0.5 mM IPTG) andsupplementation with both EGTA and IPTG to test the production andsecretion of OVA. As shown in FIG. 3B, the type III secretion system ofKBMA P. aeruginosa is functional: the antigen S54-Bla is injected intocells.

To look further about the TTSS functionality of our KBMA strain, we usedan assay measuring the cleavage of CCF2 in eukaryotic cell injected byβ-lactamase fused to ExoS54. This test of the “effective injection ofthe antigen into cells” was previously described by Derouazi et al.{Derouazi, 2010 #203}. Briefly, after 3 h of infection of eukaryoticHL60 cells with various strains, cells were incubated with freshlyprepared 6×CCF2/AM solution (1 μM final concentration; Invitrogen) for30 min in the darkness at room temperature. The percentage of cells thatreceived reporter fusions was quantified by flow cytometry (FACS Moflo;Dako Cytomation).

The five different tested strains are:

OSTAB S54Bla: Strain of P. aeruginosa known to inject S54-Bla via TTSS

OSTAB PopBD S54Bla: Strain of P. aeruginosa deleted for translocon PopBDunable to inject Bla via TTSS into cell

KBMA S54Bla: KBMA expressing S54-bla antigen

KBMA PopBD S54Bla: KBMA strain deleted for translocon PopBD

Heat killed: P. aeruginosa expressing S54-Bla heated at 70° C. for 1hour

Viable equivalent: Based on CFU graph, for KBMA at MOI 500 there isapproximately 1 bacteria alived. This is 1 bacteria OSTAB S54 Bla.

The results are shown in FIG. 3B, and are expressed as number of cellsthat exhibit a blue fluorescence; uninfected cells incubated with CCF2were used as negative control. FACS analysis revealed blue cellsindicating CCF2-AM cleavage by Bla. No blue cells were observed in cellsunstained, uninfected or infected with either equivalent viable bacteria(i.e 1 bacteria equivalent for MOI 500) or strain harbouring emptyplasmid (pEAi) or plasmid expressing wild type β-lactamase protein fusedto S54 but not secreted via TTSS (OSTABΔpopBD S54-Bla) (FIG. 2B). UsingKBMA strain, blue cells could be seen in HL60 infected at MOI 100 and500 with increased yield, indicating that the TTSS is active under PCT.Together this data demonstrate that we obtain a KBMA P. aeruginosa at 10μM S59-psoralen concentration, unable to replicate on nutrient agarmedium but still able to secrete an antigen by its activated type IIIsecretion system.

Example 5. Cytotoxicity of the Attenuated Strain Obtained in Example 3

The loss of cytotoxicity was assessed by infection of mouse dendriticcells (BMDCs). The PCT treated bacteria were prepared as mentionedabove. Cytotoxicity was assessed by determination of lactatedehydrogenase release into supernatants of the infected cells using acytotoxicity detection kit (LDH, Roche) as described previously{Dacheux, 1999 #81}. LDH release of infected cells was measured at 3 hpost-infection. The percentage of cytotoxicity for each experiment wascalculated with the following equation: (Exp. value−control with onlycells)/(control with Triton X-100 1%−control with only cells)×100. Dataare the means of results of at least three experiments.

Data are shown on FIG. 4A: photo-treated cells are not cytotoxic towardsmouse dendritic cells. FIG. 4B shows the cytotoxicity on human dendriticcells. Tested strains are the following: OSTAB and KBMA expressing M1Flu Puerto Rico antigen (M1-K252) fused to ExoS54. FIG. 4C shows theviability of human moDCs 24 h post infection with KBMA or OSTABS54-M1FI.

Example 6. Vaccination Protocol with the Adapted Strain Obtained inExample 1

Animal Experiments:

Female C57BL/6 mice were purchased from Janvier SA (LeGenest-Saint-Isle, France) and experimented at 6-8 weeks of age. Theywere kept under pathogen-free conditions in the animal facility of theUniversity Joseph Fourier (Grenoble, France). All animal experimentswere approved by the Animal Experiment Committee of the Region and wereperformed in accordance with institutional and national guidelines.

Animal Immunization:

A) Prophylactic assay: C57BL/6 mice were injected at four positions inright and left franks with CHA-CLIN1-PADRE-OVA strain at 5*10⁷cells/position/time twice 14 days and 7 days before B16OVA tumorchallenge. CHA-OST-EI is the negative control. Kaplan-Meyer curvesdisplayed survival data from groups of 6 mice. Statistical analysis:p<0.01 for three immunized groups vs EI, no difference between immunizedgroups.

B) Therapeutic assay: C57BL/6 mice received a subcutaneously tumorchallenge with GL261 tumor cells at day 0 and then were vaccinated withat four positions in right and left franks with CHA-CLIN1-PADRE-OVAstrain at 5*10⁷ cells/position/time on following D1/D5/D9/D13/D17/D21therapeutic schema. Kaplan-Meyer curves displayed survival data fromgroups of 6 mice. Statistical analysis: p<0.01 for three immunizedgroups vs EI; CHA-OST-PADRE-OVA vs CHA-CLIN1-PADRE-OVA 4 positions,p=0.72; CHA-OST-PADRE-OVA vs CHA-CLIN1-PADRE-OVA 1 position, p=0.038).

Tumor Challenge Experiment:

Immunized (prophylactic groups) or non-immunized (therapeutic groups)female C57BL/6 mice were injected subcutaneously in left flank on day 0with B16-OVA cells at the dose of 2*10⁵ cells (100 μL)/mouse or GL261cells at the dose of 1*10⁵ cells (100 μL)/mouse. The emergence and thedynamic growth of tumor mass were measured every 48 hours. When thediameter of tumor exceeded 10 mm, mice were sacrificed and this daycorresponded to the period of survival. Analysis of data was realizedwith GraphPad Prism 5 software.

Statistical Analysis:

Bar graph analyses were evaluated by Student's t-test. Kaplan-Meyersurvival curves of animals treated with different protocols wereanalyzed using the log rank test. A p-value below 0.05 between groupswas considered to indicate a statistically significant difference.

Results:

First of all, we found that 10⁸ CHA-CLIN1-PADRE-OVA/time primed thestrongest CD8+ T response among the three doses, and we tried tooptimize CHA-CLIN1 strain efficiency through a vaccination of mice atmulti-positions (four subcutaneous injections in right and left flanks,5*10⁷ bacteria/position/time, on D-14/D-7). CHA-CLIN1-PADRE-OVAvaccinations based on different protocols demonstrated the same efficacyas CHA-OST-PADRE-OVA strain (FIG. 5A).

For therapeutic assay, all immunized groups presented tumor rejectionswith slight differences between the three immunized group,CHA-CLIN1-PADRE-OVA vaccinations at four positions were slightly moreefficient than CHA-OST-PADRE-OVA vaccinations which were still betterthan CHA-CLIN1-PADRE-OVA vaccinations at one position. It has to benoticed that no significant differences were found betweenCHA-OST-PADRE-OVA group and CHA-CLIN1-PADRE-OVA 4 position group, whilethe difference between CHA-OST-PADRE-OVA group and CHA-CLIN1-PADRE-OVA 1position group was significant (p=0.038) (FIG. 5B).

CITED PATENT DOCUMENTS

-   EP 1 692 162 B1—Université Joseph Fourier

CITED NON-PATENT DOCUMENTS

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What is claimed is:
 1. A process for obtaining an adapted strain ofPseudomonas, comprising: deleting genes ExoS, ExoT, aroA and lasl in aninitial Pseudomonas strain cultivated in a LB medium; progressivelychanging the culture medium from LB medium to a chemically definedmedium comprising magnesium and calcium; and selecting the adaptedstrains presenting a doubling time inferior to 60 minutes whencultivated in the chemically defined medium.
 2. The process according toclaim 1, wherein the adapted strain is furthermore treated to become‘killed but metabolically active’.
 3. An adapted strain of Pseudomonassuch as obtained by the process according to claim 2, wherein theadapted strain presents the same toxicity and secretion capacities thanthe initial strain, and its doubling time when cultivated in thechemically defined medium is less than 60 minutes.
 4. An immunogeniccomposition comprising the adapted strain of Pseudomonas according toclaim
 3. 5. Use of an adapted strain of Pseudomonas according to claim3, for preparing a vaccine composition.